Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen that infects multiple tissue sites and medical devices. P. aeruginosa forms robust biofilms that exhibit high levels of resistance to antibiotics and are difficult to clear. In this application, we propose to test how PilZ regulates biofilm formation and dispersal. Bacteria lacking PilZ rapidly clump in liquid when [c-di-GMP] levels rise, suggesting a role for PilZ in sensing or mediating this second messenger signal?and yet the c-di-GMP binding motifs of PilZ are degenerate, and the protein does not bind cyclic-d-GMP in vitro. In Aim 1 of this proposal we will manipulate the c-di-GMP binding surface of PilZ, to determine whether PilZ interacts with c-di-GMP in vivo. We have devised a clever INSeq based strategy to identify gene products required for the rapid clumping phenotype, and present proof-of-principle data showing that the screening strategy can identify rare mutants that fail to clump. Lastly, flow cell experiments will better characterize initiation, maturation and dispersal of pilZ biofilms, and indicate whether disruption of PilZ function can be a strategy to disrupt fitness of biofilm grown bacteria.